|
| Status |
Public on May 23, 2024 |
| Title |
2C G1 Expt. 2 - Sample A3 |
| Sample type |
SRA |
| |
|
| Source name |
2-Cell Embryo
|
| Organism |
Mus musculus |
| Characteristics |
tissue: 2-Cell Embryo
treatment: Nocodazole synchronization
|
| Treatment protocol |
In Vitro Ferilized embryos were maintained in KSOM.
To obtain synchronized 2-cell stage embryos, zygotes were collected 16 hours post fertilization. Subsequently, they were incubated in a 40 nM Nocodazole solution for 4 hours, following which they were washed to facilitate release.
Mouse ES cells were sorted using FACS cytometry and prepared for RepliSeq based on a previously published protocol (PMID: 31406346).
|
| Growth protocol |
To obtain zygotes and 2-cell stage embryos, four to eight-week-old donors were injected with 100µl of 50 I.U. ml-1 (international units) ml−1 solution) solution pregnant mare serum gonadotropin (PMSG) at 17.00 followed by injection of 100µl of 50 I.U. ml-1 human chorionic gonadotropin (hCG) at 17.00 48 hours after PMSG. Mouse ES cells were cultured on 6-well plates, which had been coated with 0.01% poly-L-ornithine for 1 hours followed by 10 ng/ml-1 laminin for a further hour. The cells were maintained in 2i + LIF and passaged every 3-4 days using TrypLE dissociation solution.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA extraction for single-cell RepliSeq was conducted following established protocols as described in a previous publication (PMID: 33230331).
Library construction was carried out using the protocols established by Miura et al., 2020 (PMID: 33230331), incorporating IDT for Illumina unique dual indexes and the KAPA Hyper Prep Kit from Roche.
single cell RepliSeq
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Raw sequencing reads were trimmed, aligned and processed according to Miura et al., 2020 (PMID: 33230331).
Data processing of trimmed abd aligned reads were processed to obtain log2 fold replication timing values according to the methodology described by Miura et al. in their publication from 2020 (PMID: 33230331).
Assembly: mm10
Supplementary files format and content: BedGraph
|
| |
|
| Submission date |
Jul 14, 2023 |
| Last update date |
May 23, 2024 |
| Contact name |
Jason A Halliwell |
| E-mail(s) |
jhalliwell@sund.ku.dk
|
| Organization name |
The University of Copenhagen
|
| Street address |
Blegdamsvej 3B
|
| City |
København |
| ZIP/Postal code |
2200 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE237400 |
The establishment of a DNA replication timing program in the early embryo. |
|
| Relations |
| BioSample |
SAMN36451863 |
| SRA |
SRX21024431 |
